Vitamin K-dependent protein S (PS) is a single chain glycoprotein of 75,000 daltons molecular mass. It serves as a cofactor to activated protein C in the inactivation of factors Va and VIIIa. The concentration of PS in human plasma is approximately 25 mg/l. Protein S is found in citrated plasma at least in two forms, free PS (PS.sub.F), comprising about 40% of total PS, and bound to C4b binding protein (C4BP), comprising about 60% of total PS. C4BP is a regulatory protein of the classical pathway of the complement system. Only the free form of PS supports the cofactor activity for activated protein C. Protein S forms complexes with C4BP in the presence of Ca++ and EDTA, and the dissociation constant (Kd) for the interaction is much lower in the presence of Ca++ (6.times.10.sup.-10 M) than in the presence of EDTA (10.sup.-9 M). This low binding constant suggests that protein S circulating in blood is either completely bound to C4BP or another component that changes the equilibrium between protein S and C4BP might be involved. This third component called protein S binding protein (PSBP) was described in plasma bovine but is not found in human plasma.
The physiological relevance of protein S is demonstrated by the observed increased risk of venous thromboembolism among individuals with congenital protein S deficiency. In addition, 30% of patients exhibiting arterial thrombosis exhibit decreases in plasma PS levels. Therefore, assays for measuring the plasma levels of PS, and particularly the levels of free PS are an important tool for the clinician. The complexation of PS with C4BP removes the anticoagulation active form of PS (PS.sub.F) from the circulation.
Previous assays for measuring plasma levels of PS included the use of a pooled normal plasma as a reference standard that contained total PS comprising free (PS.sub.F) and complexed (PS:C4BP) PS. Thus the assay must separate free PS from complexed PS in order to identify the amount of PS.sub.F available in the blood. Edson et al., Am. J. Clin. Path., 94:176-186 (1990), reviews laboratory diagnostic methods for detecting free protein S, including the standard two dimensional rocket crossed immunoelectrophoresis (CIEP) procedure of Laurell et al., Anal. Biochem., 10:358-361 (1985), and the two step precipitation procedure of Comp et al., Blood, 67:504-508 (1986), using polyethylene glycol to selectively remove PS:C4BP complex from free PS prior to measurement of PS.
Antibodies immunospecific for free PS have not been described. Attempts to develop antibodies that bind the region of PS involved in binding C4BP, and that therefore would inhibit the binding of PS to C4BP have also not been successful. Dahlback et al., J. Biol. Chem., 265:8127-8135 (1990). Thus there is presently no direct means to immunologically distinguish free PS from PS:C4BP complex. Because direct assay of free PS is not presently available, assays for free PS require a separation step to distinguish the immunologically indistinguishable species of free PS from PS:C4BP complex.
Malm et al., describes a monoclonal antibody that immunoreacts with protein S (Eur. J. Biochem., 165:39-45, 1987). The antibody described binds free protein S and binds protein S complexed with C4b binding protein (C4BP), but does not bind thrombin-cleaved protein S, and is therefore proposed to bind an epitope located near the gla domain of protein S.
Recently the synthetic peptide GVQLDLDEAI (SEQ ID NO 6: 3-17) was described that is derived from the carboxy terminal region of protein S (residues 605 to 614 of mature PS) and that inhibits the interaction of protein S with C4BP in vitro. Walker et al., J. Biol. Chem., 264:17645-17648 (1989); and Weinstein et al., J. Clin Invest., 86:1928-1935 (1990). These reports suggest that this region of protein S is important for the binding to C4BP. Other protein S polypeptides corresponding to residues 608-616 and 616-624 were shown to have a measurable effect on the binding of PS to C4BP.
Additional fragments of protein S have been described in the literature that are produced by proteolytic cleavage. Dahlback et al., J. Biol. Chem., 261:5111-5115 (1986); and Stenflo et al., Natl. Acad. Sci. U.S.A., 84:368-372 (1987). However, none of these fragments have been identified as having the capacity to inhibit protein S binding to C4BP.